Attenuated live Salmonella vaccines have ability to induce antibodies cross-reactive to other enteric pathogens, particularly in strains engineered to achieve down-regulation of O-antigen synthesis in vivo. It is possible that if the Salmonella lipopolysaccharide (LPS) is truncated further, this may result in more effective presentation of conserved outer membrane proteins (OMPs) to the host immune system, which will lead to an immune response that is cross protective against a number of other Salmonella species and to other Gram- negative enteric pathogens. In addition, exposure of the fairly conserved core oligosaccharide and enhanced production of outer membrane vesicles may also aid in production of a cross-protective immune response. In this proposal, we will construct a series of mutations to achieve regulated O-antigen or core synthesis such that full length LPS is present at the time of immunization but is lost after colonization of the host. We will also include mutations that up-regulate expression of genes that specify essential ion uptake proteins including the iron regulated outer membrane proteins (IROMPs). Candidate vaccine strains will be further modified by introduction of other mutations including the pagC and ompX genes that are maximally up-regulated in vivo to promote outer membrane vesicles production to maximally induce antibodies cross-reactive to the OMPS of other enteric pathogens. The success of this project could lead to efficacious RASV with broad spectrum protection against enteric bacteria for human use. PUBLIC HEALTH RELEVANCE: Effective live bacterial vaccines hold the promise of providing low cost, orally administered, life-long protection against enteric bacteria. The goal of this project is to construct novel Salmonella vaccines to maximally expose conserved outer membrane proteins to the immune system to induce the higher cross-reactive antibodies to enteric bacteria via shutting off core oligosaccharide synthesis in vivo but retaining intact the LPS in vitro to facilitate invasion.